IL-21 antagonists

ABSTRACT

The polypeptides, and the polynucleotides encoding for them, described herein are IL-21 antagonists that bind with specificity and exhibit an EC 50  that is not detectable in receptor binding studies. These molecules have mutations in the D helix of the IL-21 molecule, and can be used to inhibit the activity of IL-21 with its cognate receptor.

REFERENCE TO RELATED APPLICATIONS

This application is related to Provisional Application No. 60/337,586,filed on Nov. 5, 2001, for which claims of benefit are made under 35U.S.C. §119(e)(1).

BACKGROUND OF THE INVENTION

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response is the T cell,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, IFN-γ, LT-α, and are involved in inflammatory responses,viral immunity, intracellular parasite immunity and allograft rejection.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and areinvolved in humoral responses, helminth immunity and allergic response.Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α.There is some evidence to suggest that Type 1 and Type 2 producing Tcell populations preferentially migrate into different types of inflamedtissue.

Mature T cells may be activated, i.e., by an antigen or other stimulus,to produce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population. Bcells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines.

The demonstrated in vivo activities of the cytokine family ininflammation and autoimmune disease illustrate the enormous clinicalpotential of, and need for cytokine antagonists. The present inventionaddresses these needs by providing antagonists of the IL-21 cytokine, aswell as related compositions and methods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INTENTION

In one aspect, the present invention provides an isolated polynucleotidemolecule comprising a nucleotide sequence as shown in SEQ ID NO: 3 orSEQ ID NO: 5. In another aspect, the present invention provides anisolated polynucleotide molecule encoding an IL-21 antagonistpolypeptide comprising an amino acid sequence as shown as in SEQ ID NO:4 or SEQ ID NO: 6. These sequences include mutations in the D helix ofIL-21.

Embodiments of the present invention include In one embodiment of thepresent invention provides an antagonist molecule with a truncation ofIL-21 polypeptide after residue 147 (Met) and wherein residue Gln₁₄₅(SEQ ID NO: 2) was mutated to an Asp_(l45) (as shown in SEQ ID NO: 4).These mutations resulted in a protein with an IC₅₀ of 10 and EC₅₀ thatwas undetectable. The resulting polypeptide, was designated zalpha11Ligand I156ST/Q153D (ST is a designation for truncation, in this case,at amino acid residue 156) shown to bind to the cognate receptor withspecificity and without any detectable signaling.

In another embodiment, the present invention provides an antagonistmolecule wherein Gln₁₄₅ (shown in SEQ ID NO: 2) has been mutated toAsp₁₄₅ (shown in SEQ ID NO: 6), and Ile₁₄₈ (shown in SEQ ID NO: 2) hasbeen mutated to Asp₁₄₈ (shown in SEQ ID NO: 6). These mutations resultedin a protein with an IC₅₀ of 10 and an EC₅₀ that was undetectable. Theresulting polypeptide, designated zalpha11 Ligand I 156D/Q153D was shownto bind to the cognate receptor with specificity and without anydetectable signaling.

One aspect of the present invention includes an isolated polypeptidecomprising an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO:6.

In another aspect, the present invention provides a fusion proteincomprising at least two polypeptides, wherein at least one of thepolypeptides comprises a polypeptide selected from the group consistingof an amino acid sequence as shown in SEQ ID NO:4 or SEQ ID NO:6, and asecond polypeptide sequence.

An other aspect of the present invention provides an expression vector,comprising the isolated nucleic acid molecule of claim 1, atranscription promoter, and a transcription terminator, wherein thepromoter is operably linked with the nucleic acid molecule, and whereinthe nucleic acid molecule is operably linked with the transcriptionterminator.

Another aspect of the present invention provides a recombinant host cellcomprising the expression vector of claim 5, wherein the host cell isselected from the group consisting of bacterium, yeast cell, fungalcell, insect cell, mammalian cell, and plant cell.

In another aspect, the present invention provides a method of producinga polypeptide, the method comprising the step of culturing recombinanthost cells that comprise the expression vector of claim 5, and thatproduce the polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within, populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequence. The term allelic variant is alsoused herein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, (α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery that certainmutations in the DNA encoding for interleukin 21 (IL-21) result inpolypeptide molecules that can bind the IL-21 receptor. In particular,mutations in the D helix IL-21 can be engineered and binding specificityretained for the IL-21 receptor. IL-21 was originally designatedzalpha11 ligand, and the receptor was originally designated zalpha11.IL-21 is described in commonly owned U.S. Pat. No. 6,307,024.

In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions. In human IL-21 amino acid sequence asshown in SEQ ID NO:2, helix A is defined by amino acid residues 41-56;helix B by amino acid residues 69-84; helix C by amino acid residues92-105; and helix D by amino acid residues 135-148. Structural analysissuggests that the A/B loop is long, the B/C loop is short and the C/Dloop is parallel long. This loop structure results in an up-up-down-downhelical organization. The cysteine residues are absolutely conservedbetween IL-21 and IL-15. The cysteine residues that are conservedbetween IL-15 and IL-21 correspond to amino acid residues 71, 78, 122and 125 of SEQ ID NO: 2. Conservation of some of the cysteine residuesis also found in IL-2, IL-4, GM-CSF and IL-21 corresponding to aminoacid residues 78 and 125 of SEQ ID NO: 2. Consistent cysteine placementis further confirmation of the four-helical-bundle structure. Alsohighly conserved in the family comprising IL-15, IL-2, IL-4, GM-CSF andIL-21 is the Glu-Phe-Leu sequence as shown in SEQ ID NO: 2 at residues136-138.

Analysis of IL-21 predicted that amino acid residues 44, 47 and 135 (asshown in SEQ ID NO: 2) played an important role in IL-21 binding to itscognate receptor. Moreover, the predicted amino acid sequence of murineIL-21 showed 57% identity to the predicted human protein. Based oncomparison between sequences of human and murine IL-21 well-conservedresidues were found in the regions predicted to encode alpha helices Aand D. The corresponding polynucleotides encoding the IL-21 polypeptideregions, domains, motifs, residues and sequences described herein are asshown in SEQ ID NO:1.

Detailed mutational analysis has been performed for IL-4 and IL-2, bothof which are highly related to IL-21. Analysis of murine IL-2 (Zurawskiet al., EMBO J. 12:5113-5119, 1993) shows residues in helices A and Care important for binding to IL-2Rβ; critical residues are Asp₃₄, Asn₉₉,and Asn₁₀₃. Multiple residues within murine IL-2 loop A/B and helix Bare important for IL-2Rα binding, while only a single residue, Gln₁₄₁ inhelix D, is vital for binding with IL-2Rα. Similarly, helices A and Care sites of interaction between IL-4 and IL-4Rα (the structurallysimilar to IL-2Rα), and residues within helix D are vital for IL-2Rαinteraction (Wang et al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997;Kruse et al., EMBO J. 11:3237-3244, 1992). In particular, the mutationTyr₁₂₄ to Asp in human IL-4 creates an antagonist, which binds withIL-4Rα but not IL-2Rα and therefore cannot signal (Kruse et al. ibid.1992).

While helix A is relatively well-conserved between human and murineIL-21, helix C is more divergent. While both species have predominantacidic amino acids in this region, the differences may account forspecies specificity in interaction between IL-21 and its “beta” typereceptor, zalpha11. Loop A/B and helix B of IL-21 are well-conservedbetween species; although no receptor subunit corresponding to IL-2Rαhas yet been identified, conservation through this region suggests thatit is functionally significant. The D helices of human and murine IL-21are also highly conserved. IL-21 receptor antagonists may be designedthrough mutations within IL-21 helix D. Any mutation which disrupts theIL-21 helical structure may abolish binding with its receptor and thusinhibit signaling.

In one embodiment of the present invention provides an antagonistmolecule with a truncation of IL-21 polypeptide after residue 147 (Met)and wherein residue Gln₁₄₅ (SEQ ID NO: 2) was mutated to an Asp₁₄₅ (asshown in SEQ ID NO: 4). These mutations resulted in a protein with anIC₅₀ of 10 and EC₅₀ that was undetectable. The resulting polypeptide,designated zalpha11 Ligand I156ST/Q153D (ST is a designation fortruncation, in this case, at amino acid residue 156) shown to bind tothe cognate receptor with specificity and without any detectablesignaling.

In another embodiment, the present invention provides an antagonistmolecule wherein Gln₁₄₅ (shown in SEQ ID NO: 2) has been mutated toAsp_(l45) (shown in SEQ ID NO: 6), and Ile₁₄₈ (shown in SEQ ID NO: 2)has been mutated to Asp₁₄₈ (shown in SEQ ID NO: 6). These mutationsresulted in a protein with an IC₅₀ of 10 and an EC₅₀ that wasundetectable. The resulting polypeptide, designated zalpha11 LigandI156D/Q153D was shown to bind to the cognate receptor with specificityand without any detectable signaling.

The functional domains of four-helical cytokines are determined on thebasis of structural homology, irrespective of sequence identity, and canmaintain functional integrity in a chimera (Kallen et al., J. Biol.Chem. 274:11859-11867, 1999). Therefore, the helical domains of IL-21antagonists will be useful for preparing chimeric fusion molecules,particularly with other short-helix form cytokines to determine andmodulate receptor binding specificity. Of particular interest are fusionproteins engineered with helix A, and fusion proteins that combinehelical and loop domains from other short-form cytokines such as IL-2,IL-4, IL-15 and GM-CSF. The amino acid residues comprising helices A, B,C, and D, and loops A/B, B/C and C/D for IL-21, IL-2, IL-4, IL-15 andGM-CSF are shown in Table 1.

TABLE 1 Helix A/B Helix B/C Helix C/D Helix A Loop B Loop C Loop D IL-2141-56 57-68 69-84  85-91  92-105 106-134 135-148 SEQ residues ID NO:2IL-2 36-46 47-52 53-75  76-86  87-99 100-102 103-121 SEQ residues ID NO:7 IL-4 29-43 44-64 65-83  84-94  95-118 119-133 134-151 SEQ residues IDNO: 8 IL-15 45-68 69-83 84-101 102-106 107-119 120-133 134-160 SEQresidues ID NO: 9 GM- 30-44 45-71 72-81  82-90  91-102 103-119 120-131SEQ CSF ID residues NO: 10

The IL-21 receptor is a member of the Class I cytokine receptorsubfamily that includes the receptors for IL-2, IL-4, IL-7, IL-15, EPO,TPO, GM-CSF and G-CSF (for a review see, Cosman, “The HematopoietinReceptor Superfamily” in Cytokine 5(2): 95-106, 1993). The IL-21receptor is fully described in commonly-owned PCT Patent Application No.US99/22149. IL-21 receptor is expressed in lymph node, peripheral bloodleukocytes (PBLs), spleen, bone marrow, and thymus. The tissuedistribution of the receptor suggests that a target for the predictedIL-21 is hematopoietic lineage cells, in particular lymphoid progenitorcells and lymphoid cells. Other known four-helical-bundle cytokines thatact on lymphoid cells include IL-2, IL-4, IL-7, and IL-15. For a reviewof four-helical-bundle cytokines, see, Nicola et al., Advances inProtein Chemistry 52:1-65, 1999 and Kelso, A., Immunol. Cell Biol.76:300-317, 1998.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the IL-21 antagonist polypeptides asdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate DNA sequence that encompasses all DNAs that encode theIL-21 polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:3 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, IL-21polypeptide-encoding polynucleotides comprising nucleotide 1 or 97 tonucleotide 486 of SEQ ID NO:3 and their RNA equivalents are contemplatedby the present invention. Table 2 sets forth the one-letter codes usedwithin SEQ ID NO:3 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, withA being complementary to T, and G being complementary to C.

TABLE 2 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 3.

TABLE 3 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

Using isolated polynucleotides of the present invention that include DNAand RNA, the native sequence of IL-21 is isolated for a template toidentify mutants. Methods for preparing DNA and RNA are well known inthe art.

The present invention also provides IL-21 antagonist polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NOS:4 or 6. The term “substantially similar sequence identity” isused herein to denote polypeptides comprising at least greater than 95%sequence identity to the sequences shown in SEQ ID NOS:4 or 6. Thepresent invention further includes nucleic acid molecules that encodesuch polypeptides. Methods for determining percent identity aredescribed below.

The present invention also contemplates variant IL-21 nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NOS:4 or 6, and/or a hybridization assay. Such IL-21variants include nucleic acid molecules: (1) that hybridize with anucleic acid molecule having the nucleotide sequence of SEQ ID NOS:3 or5 (or their complements) under stringent washing conditions, in whichthe wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65°C.; or (2) that encode a polypeptide having at least greater than 95%sequence identity to the amino acid sequence of SEQ ID NOS:4 or 6.Alternatively, IL-21 antagonists can be characterized as nucleic acidmolecules: (1) that hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NOS:3 or 5 (or their complements) underhighly stringent washing conditions, in which the wash stringency isequivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) thatencode a polypeptide having at least greater than 95% sequence identityto the amino acid sequence of SEQ ID NOS:4 or 6.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 4 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{matrix}\left\lbrack {{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \right. \\\begin{matrix}{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\\left. {{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}} \right\rbrack\end{matrix}\end{matrix}} \times 100$

TABLE 4 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant IL-21. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NOS:4 or 6) and atest sequence that have either the highest density of identities (if thektup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant IL-21 polypeptides or polypeptides with substantially similarsequence identity are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table5) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue, a small linker peptide of up toabout 20-25 residues, or an affinity tag. The present invention thusincludes polypeptides of from about 108 to 216 amino acid residues thatcomprise a sequence that is at least 95% or more identical to thecorresponding region of SEQ ID NOS:4 or 6. Polypeptides comprisingaffinity tags can further comprise a proteolytic cleavage site betweenthe IL-21 polypeptide and the affinity tag. Preferred such sites includethrombin cleavage sites and factor Xa cleavage sites.

TABLE 5 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules. The effects of amino acid sequencechanges can be predicted by, for example, computer modeling as disclosedabove or determined by analysis of crystal structure (see, e.g.,Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other techniquesthat are well known in the art compare folding of a variant protein to astandard molecule (e.g., the native protein). For example, comparison ofthe cysteine pattern in a variant and standard molecules can be made.Mass spectrometry and chemical modification using reduction andalkylation provide methods for determining cysteine residues which areassociated with disulfide bonds or are free of such associations (Beanet al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci.2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732,1994). It is generally believed that if a modified molecule does nothave the same cysteine pattern as the standard molecule folding would beaffected. Another well known and accepted method for measuring foldingis circular dichrosism (CD). Measuring and comparing the CD spectragenerated by a modified molecule and standard molecule is routine(Johnson, Proteins 7:205-214, 1990). Crystallography is another wellknown method for analyzing folding and structure. Nuclear magneticresonance (NMR), digestive peptide mapping and epitope mapping are alsoknown methods for analyzing folding and structurally similaritiesbetween proteins and polypeptides (Schaanan et al., Science 257:961-964,1992).

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes aIL-21 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NOS:3 or 5 or fragments thereof, can bedigested with Bal31 nuclease to obtain a series of nested deletions.These DNA fragments are then inserted into expression vectors in properreading frame, and the expressed polypeptides are isolated and testedfor IL-21 activity, or for the ability to bind anti-IL-21 antibodies orIL-21 receptor. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired IL-21 fragment. Alternatively,particular fragments of a IL-21 gene can be synthesized using thepolymerase chain reaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1 Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204), and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed IL-21 nucleotide and polypeptide sequences canalso be generated through DNA shuffling as disclosed by Stemmer, Nature370:389 (1994), Stemmer, Proc. Natl Acad. Sci. USA 91:10747 (1994), andinternational publication No. WO 97/20078. Briefly, variant DNAmolecules are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-IL-21 antibodies or soluble IL-21 receptor, can berecovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly other cytokines, to provide multi-functional molecules. Forexample, one or more helices from IL-21 can be joined to other cytokinesto enhance their biological properties or efficiency of production.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the helices of IL-21 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is typically carried out in acell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993). It may be advantageous tostabilize IL-21 to extend the half-life of the molecule, particularlyfor extending metabolic persistence in an active state. To achieveextended half-life, IL-21 molecules can be chemically modified usingmethods described herein. PEGylation is one method commonly used thathas been demonstrated to increase plasma half-life, increasedsolubility, and decreased antigenicity and immunogenicity (Nucci et al.,Advanced Drug Delivery Reviews 6:133-155, 1991 and Lu et al., Int. J.Peptide Protein Res. 43:127-138, 1994).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for IL-21 amino acidresidues.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a IL-21 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

Regardless of the particular nucleotide sequence of a IL-21 antagonistpolynucleotide, the polynucleotide encodes a polypeptide that ischaracterized by its ability to induce or inhibit specialized cellfunctions, or by the ability to bind specifically to an anti-IL-21antibody or Il-21 receptor. More specifically, an IL-21 antagonist willbind its cognate receptor with at least an IC₅₀ of 100 nM and exhibit anEC₅₀ of 100 nM or greater.

For any IL-21 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above.

The present invention further provides a variety of other polypeptidefusions (and related multimeric proteins comprising one or morepolypeptide fusions). For example, a IL-21 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-IL-21polypeptide fusions can be expressed in genetically engineered cells (toproduce a variety of multimeric IL-21 analogs). Auxiliary domains can befused to IL-21 polypeptides to target them to specific cells, tissues,or macromolecules. For example, a IL-21 polypeptide or protein could betargeted to a predetermined cell type by fusing a IL-21 polypeptide to aligand that specifically binds to a receptor on the surface of thattarget cell. In this way, polypeptides and proteins can be targeted fortherapeutic or diagnostic purposes. A IL-21 polypeptide can be fused totwo or more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9, 1996.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that havesubstantially similar sequence identity to residues 1-162 or 33-162 ofSEQ ID NOS: 4 or 6, wherein such polypeptides or fusions bind thezalpha11 receptor or IL-21 antibodies with an IC₅₀ of 100 nM or less,and exhibit an EC₅₀ of 100 nM or greater.

The IL-21 polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion polypeptides, can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells.Eukaryotic cells, particularly cultured cells of multicellularorganisms, are preferred. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a IL-21 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a IL-21 polypeptide into the secretory pathway of a host cell,a secretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of zalpha11 Ligand, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is operably linked to the IL-21 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residue 1-31of SEQ ID NO:2 is be operably linked to a DNA sequence encoding anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the IL-21 polypeptide into a baculovirus genomemaintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case zalpha11Ligand. However, pFastBac1™ can be modified to a considerable degree.The polyhedrin promoter can be removed and substituted with thebaculovirus basic protein promoter (also known as Pcor, p6.9 or MPpromoter) which is expressed earlier in the baculovirus infection, andhas been shown to be advantageous for expressing secreted proteins. See,Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol. 71: 971-6, 1990;Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk,G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native IL-21 secretory signal sequences with secretorysignal sequences derived from insect proteins. For example, a secretorysignal sequence from Ecdysteroid Glucosyltransferase (EGT), honey beeMelittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative IL-21 secretory signal sequence. In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed IL-21 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci.82:7952-4, 1985). Using techniques known in the art, a transfer vectorcontaining IL-21 is transformed into E. Coli, and screened for bacmidswhich contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirus isisolated, using common techniques, and used to transfect Spodopterafrugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses IL-21is subsequently produced. Recombinant viral stocks are made by methodscommonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillernondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (Ω) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a IL-21polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant IL-21 polypeptides (or chimeric IL-21polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their physical or biochemical properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins, including those comprising polyhistidinetags. Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39) and use of the solublezalpha11 receptor. Within additional embodiments of the invention, afusion of the polypeptide of interest and an affinity tag (e.g.,maltose-binding protein, an immunoglobulin domain) may be constructed tofacilitate purification.

IL-21 polypeptides or fragments thereof may also be prepared throughchemical synthesis. IL-21 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue. For example,the polypeptides can be prepared by solid phase peptide synthesis, forexample as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963.

The activity of molecules of the present invention can be measured usinga variety of assays that measure proliferation of and/or binding tocells expressing the IL-21 receptor. Of particular interest are changesin IL-21-dependent cells. Suitable cell lines to be engineered to beIL-21-dependent include the IL-3-dependent BaF3 cell line (Palacios andSteinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell.Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790,1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growthfactor-dependent cell lines can be established according to publishedmethods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexteret al., in Baum et al. Eds., Experimental Hematology Today, 8th Ann.Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).

Proteins of the present invention are useful for induction or inhibitionof specialized cell functions of cells of the hematopoietic lineages,including, but not limited to, T cells, B cells, NK cells, dendriticcells, monocytes, and macrophages, as well as epithelial cells. IL-21antagonists are used to block IL-21 binding and signal transduction invitro and in vivo. These anti-IL-21 binding polypeptides would be usefulfor inhibiting IL-21 activity or protein-binding. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990, incorporated herein by reference), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989, incorporated herein by reference), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference). Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

The molecules of the present invention can be assayed in vivo usingviral delivery systems. Exemplary viruses for this purpose includeadenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997).

In view of the tissue distribution observed for IL-21 receptor agonists(including the natural Il-21/substrate/ cofactor/etc.) and/orantagonists have enormous potential in both in vitro and in vivoapplications. Antagonists are useful as research reagents forcharacterizing sites of ligand-receptor interaction. Antagonists areuseful to inhibit expansion, proliferation, activation, and/ordifferentiation of cells involved in regulating hematopoiesis.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.Alternatively, ligand/receptor binding can be analyzed using SELDI(TM)technology (Ciphergen, Inc., Palo Alto, Calif.).

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

IL-21 antagonist polypeptides can also be used to prepare antibodiesthat bind to IL-21 epitopes, peptides or polypeptides. The IL-21polypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. One of skill in theart would recognize that antigenic, epitope-bearing polypeptides containa sequence of at least 6, preferably at least 9, and more preferably atleast 15 to about 30 contiguous amino acid residues of a IL-21polypeptide (e.g., SEQ ID NOS:4 or 6). Polypeptides comprising a largerportion of IL-21 antagonist polypeptide, i.e., from 30 to 100 residuesup to the entire length of the amino acid sequence are included.Antigens or immunogenic epitopes can also include attached tags,adjuvants and carriers, as described herein. Suitable antigens includethe full length and the mature IL-21 antagonist polypeptide, mutanthelix D, as described herein.

IL-21 antagonist fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood and bone marrow cancers),if the IL-21 polypeptide or anti-IL-21 antibody targets thehyperproliferative blood or bone marrow cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). The described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable IL-21 polypeptidesor anti-IL-21 antibodies target an undesirable cell or tissue (i.e., atumor or a leukemia), and the fused cytokine mediated improved targetcell lysis by effector cells. Suitable cytokines for this purposeinclude interleukin 2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), for instance.

IL-21 was isolated from tissue known to have important immunologicalfunction and which contain cells that play a role in the immune system.IL-21 is expressed in CD3+ selected, activated peripheral blood cells,and it has been shown that IL-21 expression increases after T cellactivation. Moreover, results of experiments have previouslydemonstrated that IL-21 polypeptides have an effect on thegrowth/expansion and/or differentiated state of NK cells or NKprogenitors. Additional evidence demonstrated that IL-21 affectedproliferation and/or differentiation of T cells and B cells in vivo.Factors that both stimulate proliferation of hematopoietic progenitorsand activate mature cells are generally known. Thus, IL-21 antagonistpolypeptides will be useful for inhibiting the growth anddifferentiation of these IL-21-responsive cell types. This isparticularly useful when proliferation of a specific responsive celltype is associated with a hyper-proliferative disease, such as cancer orautoimmune disease.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference). Alternatively, IL-21 polypeptide itself can serveas an additional cell-surface or secreted marker associated withstage-specific expression of a tissue. As such, direct measurement ofIL-21 polypeptide, or its loss of expression in a tissue as itdifferentiates, can serve as a marker for differentiation of tissues.

The activity and effect of IL-21 antagonists on tumor progression andmetastasis can be measured in vivo. Several syngeneic mouse models havebeen developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6/J mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6/J mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79:315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing IL-21, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., IL-21, on theability of the tumor to recruit vasculature and undergo metastasis canthus be assessed. In addition, aside from using adenovirus, theimplanted cells can be transiently transfected with IL-21. Use of stableIL-21 transfectants as well as use of induceable promoters to activateIL-21 expression in vivo are known in the art and can be used in thissystem to assess IL-21 induction of metastasis. Moreover, purified IL-21or IL-21 conditioned media can be directly injected in to this mousemodel, and hence be used in this system. For general reference see,O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. MurineModels of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

IL-21 antagonists could be administered in combination with other agentsalready in use including both conventional chemotherapeutic agents aswell as immune modulators such as interferon alpha. Alpha/betainterferons have been shown to be effective in treating some leukemiasand animal disease models, and the growth inhibitory effects ofinterferon-alpha and IL-21 are additive for at least one B-celltumor-derived cell line.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition of IL-21antagonist sufficient to reducing proliferation of the neoplastic B or Tcells. In other embodiments, the composition can comprise at least oneother cytokine selected from the group consisting of IL-2, IL-15, IL-4,GM-CSF, Flt3 ligand or stem cell factor. Furthermore, the Il-21antagonist can be a ligand/toxin fusion protein.

The tissue distribution of a receptor for a given cytokine offers astrong indication of the potential sites of action of that cytokine.Northern analysis of IL-21 receptor revealed transcripts in humanspleen, thymus, lymph node, bone marrow, and peripheral bloodleukocytes. Specific cell types were identified as expressing zalpha11receptors, and strong signals were seen in a mixed lymphocyte reaction(MLR) and in the Burkitt's lymphoma Raji. The two monocytic cell lines,THP-1 (Tsuchiya et al., Int. J. Cancer 26:171-176, 1980) and U937(Sundstrom et al., Int. J. Cancer 17:565-577, 1976), were negative.

IL-21 receptor is expressed at relatively high levels in the MLR, inwhich peripheral blood mononuclear cells (PBMNC) from two individualsare mixed, resulting in mutual activation. Detection of high levels oftranscript in the MLR but not in resting T or B cell populationssuggests that Il-21 receptor expression may be induced in one or morecell types during activation. Activation of isolated populations of Tand B cells can be artificially achieved by stimulating cells with PMAand ionomycin. When sorted cells were subjected to these activationconditions, levels of IL-21 receptor transcript increased in both celltypes, supporting a role for this receptor and IL-21 in immuneresponses, especially in autocrine and paracrine T and B cell expansionsduring activation. IL-21 may also play a role in the expansion of moreprimitive progenitors involved in lymphopoiesis.

IL-21 receptor was found to be present at low levels in resting T and Bcells, and was upregulated during activation in both cell types.Interestingly, the B cells also down-regulate the message more quicklythan do T cells, suggesting that amplitude of signal and timing ofquenching of signal are important for the appropriate regulation of Bcell responses.

In addition, a large proportion of intestinal lamina propria cells showpositive hybridization signals with IL-21 receptor. This tissue consistsof a mixed population of lymphoid cells, including activated CD4+ Tcells and activated B cells. Immune dysfunction, in particular chronicactivation of the mucosal immune response, plays an important role inthe etiology of Crohn's disease; abnormal response to and/or productionof proinflammatory cytokines is also a suspected factor (Braegger etal., Annals Allergy 72:135-141, 1994; Sartor R B Am. J. Gastroenterol.92:5S-11S, 1997. IL-21 in concert with IL-15 expands NK cells from bonemarrow progenitors and augments NK cell effector function. IL-21 alsoco-stimulates mature B cells stimulated with anti-CD40 antibodies, butinhibits B cell proliferation to signals through IgM. IL-21 enhances Tcell proliferation in concert with a signal through the T cell receptor,and overexpression in transgenic mice leads to lymphopenia and anexpansion of monocytes and granulocytes. These pleiotropic effects ofIL-21 suggest that it can provide therapeutic utility for a wide rangeof diseases arising from defects in the immune system, including (butnot limited to) systemic lupus erythematosus (SLE), rheumatoid arthritis(RA), multiple sclerosis (MS), myasthenia gravis, and diabetes. It isimportant to note that these diseases are the result of a complexnetwork of immune dysfunction (SLE, for example, is the manifestation ofdefects in both T and B cells), and that immune cells are dependent uponinteraction with one another to elicit a potent immune response.Therefore, IL-21 antagonist that can be used to manipulate more than onetype of immune cell is an attractive therapeutic candidate forintervention at multiple stages of disease.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. The bioactive polypeptide orantibody conjugates described herein can be delivered intravenously,intraarterially or intraductally, or may be introduced locally at theintended site of action. Intravenous administration will be by bolusinjection or infusion over a typical period of one to several hours. Ingeneral, pharmaceutical formulations will include a IL-21 protein incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doseswill generally be in the range of 0.1 to 100 μg/kg of patient weight perday, preferably 0.5-20 μg/kg per day, with the exact dose determined bythe clinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of IL-21 is an amount sufficient to produce aclinically significant change in hematopoietic or immune function.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Construction of MPL-IL-21 Receptor PolypeptideChimera: MPL Extracellular and TM Domain Fused to the IL-21 ReceptorIntracellular Signaling Domain

The extracellular and transmembrane domains of the murine MPL receptorwere isolated from a plasmid containing the murine MPL receptor(PHZ1/MPL plasmid) using PCR with primers ZC17,212 (SEQ ID NO:11) andZC19,914 (SEQ ID NO:12). The reaction conditions were as follows: 95° C.for 1 min.; 35 cycles at 95° C. for 1 min., 45° C. for 1 min., 72° C.for 2 min.; followed by 72° C. at 10 min.; then a 10° C. soak. The PCRproduct was run on a 1% low melting point agarose (Boerhinger Mannheim,Indianapolis, Ind.) and the approximately 1.5 kb MPL receptor fragmentisolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions.

The intracellular domains of human IL-21 receptor were isolated from aplasmid containing IL-21 receptor cDNA using PCR with primers ZC19,913(SEQ ID NO:13) and ZC20,097 (SEQ ID NO:14). The polynucleotide sequencecorresponding to the IL-21 receptor coding sequence is shown in SEQ IDNO:15, and the corresponding amino acid sequence shown in SEQ ID NO:16.The reaction conditions were as per above. The PCR product was run on a1% low melting point agarose (Boerhinger Mannheim) and the approximately900 bp IL-21 receptor fragment isolated using Qiaquick gel extractionkit as per manufacturer's instructions.

Each of the isolated fragments described above were mixed at a 1:1volumetric ratio and used in a PCR reaction using ZC17,212 (SEQ IDNO:11) and ZC20,097 (SEQ ID NO:14) to create the MPL-IL-21 receptorchimera. The reaction conditions were as follows: 95° C. for 1 min.; 35cycles at 95° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.;followed by 72° C. at 10 min.; then a 10° C. soak. The entire PCRproduct was run on a 1% low melting point agarose (Boehringer Mannheim)and the approximately 2.4 kb MPL-IL-21 receptor chimera fragmentisolated using Qiaquick gel extraction kit (Qiagen) as permanufacturer's instructions. The MPL-IL-21 receptor chimera fragment wasdigested with EcoRI (BRL) and XbaI (Boerhinger Mannheim) as permanufacturer's instructions. The entire digest was run on a 1% lowmelting point agarose (Boehringer Mannheim) and the cleaved MPL-IL-21receptor chimera isolated using Qiaquick™ gel extraction kit (Qiagen) asper manufacturer's instructions. The resultant cleaved MPL-IL-21receptor chimera was inserted into an expression vector as describedbelow.

Recipient expression vector pZP-5N was digested with EcoRI (BRL) andHindIII (BRL) as per manufacturer's instructions, and gel purified asdescribed above. This vector fragment was combined with the EcoRI andXbaI cleaved MPL-IL-21 receptor chimera isolated above and aXbaI/HindIII linker fragment in a ligation reaction. The ligation wasrun using T4 Ligase (BRL), at 15° C. overnight. A sample of the ligationwas electroporated in to DH10B ElectroMAX™ electrocompetent E. colicells (25 μF, 200Ω, 2.3V). Transformants were plated on LB+Ampicillinplates and single colonies screened by PCR to check for the MPL-IL-21receptor chimera using ZC17,212 (SEQ ID NO:11) and ZC20,097 (SEQ IDNO:14) using the PCR conditions as described above.

Example 2 MPL-IL-21 Receptor Chimera-Based Proliferation in BAF3 AssayUsing Alamar Blue

A. Construction of BaF3 Cells Expressing MPL-IL-21 Receptor Chimera

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, pZP-5N/MPL-IL-21 receptorplasmid DNA (Example 1) was prepared and purified using a Qiagen MaxiPrep kit (Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed once in RPMI media and then resuspended inRPMI media at a cell density of 10⁷ cells/ml. One ml of resuspended BaF3cells was mixed with 30 μg of the pZP-5N/MPL-IL-21 receptor plasmid DNAand transferred to separate disposable electroporation chambers (GIBCOBRL). Following a 15 minute incubation at room temperature the cellswere given two serial shocks (800 lFad/300 V.; 1180 lFad/300 V.)delivered by an electroporation apparatus (CELL-PORATOR™; GIBCO BRL).After a 5 minute recovery time, the electroporated cells weretransferred to 50 ml of complete media and placed in an incubator for15-24 hours (37° C., 5% CO₂). The cells were then spun down andresuspended in 50 ml of complete media containing Geneticin™ (Gibco)selection (500 μg/ml G418) in a T-162 flask to isolate theG418-resistant pool. Pools of the transfected BaF3 cells, hereinaftercalled BaF3/MPL-IL-21 receptor cells, were assayed for signalingcapability as described below.

Example 3 Construction of Expression Vector Expressing Full-Length IL-21Receptor

The entire IL-21 receptor was isolated from a plasmid containingzalpha11 receptor cDNA (SEQ ID NO:15) using PCR with primers ZC19,905(SEQ ID NO:19) and ZC19,906 (SEQ ID NO:20). The reaction conditions wereas follows: 95° C. for 1 min; 35 cycles at 95° C. for 1 min, 55° C. for1 min, 72° C. for 2 min; followed by 72° C. at 10 min; then a 10° C.soak. The PCR product was run on a 1% low melting point agarose(Boerhinger Mannheim) gel and the approximately 1.5 kb zalpha11 cDNAisolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions.

The purified IL-21 receptor cDNA was digested with BamHI (BoerhingerMannheim) and EcoRI (BRL) as per manufacturer's instructions. The entiredigest was run on a 1% low melting point agarose (Boerhinger Mannheim)gel and the cleaved IL-21 receptor fragment was purified the usingQiaquick™ gel extraction kit as per manufacturer's instructions. Theresultant cleaved IL-21 receptor fragment was inserted into anexpression vector as described below.

Recipient expression vector pZP-5N was digested with BamHI (BoerhingerMannheim) and EcoRI (BRL) as per manufacturer's instructions, and gelpurified as described above. This vector fragment was combined with theBamHI and EcoRI cleaved IL-21 receptor fragment isolated above in aligation reaction using T4 Ligase (BRL). The ligation was incubated at15° C. overnight. A sample of the ligation was electroporated in toDH10B electroMAX™ electrocompetent E. Coli cells (25 μF, 200Ω, 2.3V).Transformants were plated on LB+Ampicillin plates and single coloniesscreened by PCR to check for the IL-21 receptor sequence using ZC19,905(SEQ ID NO:19) and ZC19,906 (SEQ ID NO:20) using the PCR conditions asdescribed above.

Example 4 IL-21 Activates Human IL-21 Receptor in Luciferase Assay

A. Construction of BaF3/KZ134/IL-21 Receptor Cell Line

The KZ134 plasmid was constructed with complementary oligonucleotidesZC12,749 (SEQ ID NO:17) and ZC12,748 (SEQ ID NO:18) that contain STATtranscription factor binding elements from 4 genes. A modified c-fos Sisinducible element (m67SIE, or hSIE) (Sadowski, H. et al., Science261:1739-1744, 1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. etal., Science 272:719-722, 1996), the mammary gland response element ofthe β-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.11:3745-3755, 1991), and a STAT inducible element of the Fcg RI gene,(Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045, 1995). Theseoligonucleotides contain Asp718-XhoI compatible ends and were ligated,using standard methods, into a recipient firefly luciferase reportervector with a c-fos promoter (Poulsen, L. K. et al., J. Biol. Chem.273:6229-6232, 1998) digested with the same enzymes and containing aneomycin selectable marker. The KZ134 plasmid was used to stablytransfect BaF3 cells, using standard transfection and selection methods,to make the BaF3/KZ134 cell line.

A stable BaF3/KZ134 indicator cell line, expressing the full-lengthIL-21 receptor was constructed as per Example 2, using about 30 μg ofthe Il-21 receptor expression vector, described in Example 3. Cloneswere diluted, plated and selected using standard techniques. Clones werescreened by luciferase assay using the human IL-21 conditioned media asan inducer. Clones with the highest luciferase response (via STATluciferase) and the lowest background were selected. A stabletransfectant cell line was selected. The cell line was calledBaF3/KZ134/IL-21 receptor.

B. Human and Mouse IL-21 Activates Human IL-21 Receptor inBaF3/KZ134/IL-21 Receptor Luciferase Assay

BaF3/KZ134/IL-21 receptor cells were spun down and washed in mouse IL-3free media. The cells were spun and washed 3 times to ensure removal ofmouse IL-3. Cells were then counted in a hemacytometer. Cells wereplated in a 96-well format at about 30,000 cells per well in a volume of100 μl per well using the mouse IL-3 free media. The same procedure wasused for untransfected BaF3/KZ134 cells for use as a control in thesubsequent assay.

STAT activation of the BaF3/KZ134/IL-21 receptor cells was assessedusing conditioned media from (1) BHK570 cells transfected with the humanIL-21 receptor or (2) BHK570 cells transfected with the mouse IL-21receptor, or (4) mIL-3 free media to measure media-only controlresponse. Conditioned media was diluted with RPMI mIL-3 free media to50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations.100 μl of the diluted conditioned media was added to theBaF3/KZ134/IL-21 receptor cells. The assay using the conditioned mediawas done in parallel on untransfected BaF3/KZ134 cells as a control. Thetotal assay volume was 200 μl. The assay plates were incubated at 37°C., 5% CO₂ for 24 hours at which time the cells were pelleted bycentrifugation at 2000 rpm for 10 min., and the media was aspirated and25 μl of lysis buffer (Promega) was added. After 10 minutes at roomtemperature, the plates were measured for activation of the STATreporter construct by reading them on a luminometer (LabsystemsLuminoskan, model RS) which added 40 μl of luciferase assay substrate(Promega) at a five second integration.

Results confirmed the STAT reporter response of the BaF3/KZ134/IL-21receptor cells to the human IL-21. The response, as measured, wasapproximately 50 fold over media-only control at the 50% concentration.STAT activation in response to human IL-21 was absent in theuntransfected BaF3/KZ134 control cells, showing that the response ismediated through the IL-21 receptor.

Results also confirmed the STAT reporter response of theBaF3/KZ134/IL-21 receptor cells to the mouse IL-21. The response, asmeasured, was approximately 40 fold over media-only control at the 50%concentration. Moreover, STAT activation in response to mouse IL-21 wasevident (about 5-fold) on the untransfected BaF/KZ134 control cells,suggesting that the murine BaF3 cells may have endogenous mousereceptor.

Example 5 Expression Vector Construction, Expression and Purification ofUntagged Human and Murine IL-21 from Baculovirus.

A. Construct for Expressing Human IL-21 in Baculovirus

An expression vector, pzalpha11L, was prepared to express Human IL-21polypeptides in insect cells. A 517 bp fragment containing sequence forHuman IL-21 and encoded BamH1 and XhoI restriction sites on the 5′ and3′ ends respectively, was generated by PCR amplification from a plasmidcontaining human IL-21 cDNA using primers ZC23,444 (SEQ ID NO:21) andZC23,445 (SEQ ID NO:22). The PCR reaction conditions were as follows: 1cycle of 94C for 4 minutes, followed by 25 cycles of 94° C. for 45seconds, 50° C. for 45 seconds, and 72° C. for 2 minutes; 1 cycle at 72°C. for 10 min; followed by a 4° C. soak. The fragment was visualized bygel electrophoresis (1% SeaPlaque/1% NuSieve). The band was excised,diluted to 0.5% agarose with 2 mM MgCl₂, melted at 65° C., digested withBamH1 and XhoI (Boerhinger Mannheim), and ligated into an BamH1/XhoIdigested baculovirus expression vector, pZBV3L. The pZBV3L vector is amodification of the pFastBac1™ (Life Technologies) expression vector,where the polyhedron promoter has been removed and replaced with thelate activating Basic Protein Promoter. About 14 nanograms of therestriction digested IL-21 insert and about 40 ng of the correspondingvector were ligated overnight at 16° C.

The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH 7.5 and 1mM EDTA) and about 4 fmol of the diluted ligation mix was transformedinto DH5α Library Efficiency competent cells (Life Technologies)according to manufacturer's direction by heat shock for 45 seconds in a42° C. waterbath. The transformed DNA and cells were diluted in 450 μlof SOC media (2% Bacto™ Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1MNaCl, 1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and platedonto LB plates containing 100 μg/ml ampicillin. Clones were analyzed byrestriction digests and 1 μl of the positive clone was transformed into20 μl DH10Bac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg,Md.) according to manufacturer's instruction, by heat shock as describedabove. The transformed cells were then diluted in 980 μl SOC media (2%Bacto™ Tryptone, 0.5% Bacto™ Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl,10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) out grown in shakingincubator at 37° C. for four hours and plated onto Luria Agar platescontaining 50 μg/ml kanamycin, 7 μg/ml gentamicin (Life Technologies),10 μg/ml tetracycline, IPTG (Pharmacia Biotech) and Bluo-Gal (LifeTechnologies). The plated cells were incubated for 48 hours at 37° C. Acolor selection was used to identify those cells having Human IL-21encoding donor insert that had incorporated into the plasmid (referredto as a “bacmid”). Those colonies, which were white in color, werepicked for analysis. Human IL-21 Bacmid DNA was isolated from positivecolonies using the QiaVac Miniprep8 system (Qiagen) according themanufacturer's directions. Clones were screened for the correct insertby amplifying DNA using primers to the transposable element in thebacmid via PCR using primers ZC447 (SEQ ID NO:23) and ZC976 (SEQ IDNO:24). The PCR reaction conditions were as follows: 35 cycles of 94° C.for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5 minutes; 1 cycleat 72° C. for 10 min.; followed by 4C soak. The PCR product was run on a1% agarose gel to check the insert size. Those clones having the correctinsert were used to transfect Spodoptera frugiperda (Sf9) cells.

B. Expression and Generation of Material for Purification of Human IL-21from Baculovirus

Sf9 cells were seeded at 5×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. Five microliters of human IL-21 bacmid DNA(above) was diluted with 100 μl Sf-900 II SFM (Life Technologies). Sixμl of CellFECTIN Reagent (Life Technologies) was diluted with 100 μlSf-900 II SFM. The bacmid DNA and lipid solutions were gently mixed andincubated 30-45 minutes at room temperature. The media from one plate ofcells were aspirated, the cells were washed 1× with 2 ml fresh Sf-900 IISFM media. Eight hundred microliters of Sf-900 II SFM was added to thelipid-DNA mixture. The wash media was aspirated and the DNA-lipid mixadded to the cells. The cells were incubated at 27° C. for 4-5 hours.The DNA-lipid mix was aspirated and 2 ml of Sf-900 II media was added toeach plate. The plates were incubated at 27° C., 90% humidity, for 96hours after which the virus was harvested.

For Primary Amplification Sf9 cells were grown in 50 ml Sf-900 II SFM ina 125 ml shake flask to an approximate density of 0.41-0.52×10⁵cells/ml. They were then infected with 150 μl of the virus stock fromabove and incubated at 27° C. for 3 days after which time the virus washarvested according to standard methods known in the art. A 500 μlsample submitted for activity in a BaF3 assay to show that it wasbiologically active.

For Secondary Amplification Sf9 cells were grown in 1L of Sf-900 II SFMin a 2800 ml shake flask to an approximate density of 0.5×10⁵ cells/ml.It was infected with 500 ul of the Primary viral stock from above andincubated at 27° C. for 4 days after which time the virus was harvestedaccording to standard methods known in the art. Virus was titered andgrown up large scale for purification of the baculovirus-produced humanIL-21 (huzalpha11L-Bv).

C. Large-scaled Purification of Baculovirus Expressed Human/Murine IL-21

Unless otherwise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying human IL-21 (huzalpha11L-Bv)from BV conditioned media. Conditioned media (CM) was sterile filteredthrough 0.45 and 0.22 micron filters, then buffered with 0.01 M MES(Fluka BioChemika, Switzerland)) and the pH adjusted to 6.0 The CM wasthen loaded onto a POROS 50 HS column and run, fractions collected andanalyzed.

The above peak fractions were pooled, concentrated run on a highresolution size exclusion column, and analyzed.

The fractions of interest from the size exclusion column were pooled andconcentrated with 5 kD MWCO Millipore centrifugal spin concentrators toa minimal volume. The final product was then analyzed by SDS-PAGECoomassie (Sigma, St. Louis, Mo.), Western immunological blotting,N-terminal sequencing, Amino Acid Analysis, and CB (Pierce, Rockford,Ill.) for protein purity and concentration as described in Example 29A.Bulk protein was stored at −80° C.

D. Small Scale (<2 mg) Purification of Baculovirus-expressedHuman/Murine IL-21

Unless other wise stated, all operations were carried out at 4° C. Thefollowing procedure was used for purifying<2 mg of human or murine IL-21from BV conditioned media. The CM was filtered, buffered and pH adjustedas in Example 30C. The CM was then loaded, eluted and the POROS 50 HSchromatography was analyzed as in Example 30C.

Fractions were pooled then concentrated via diafiltration in a stirredcell concentrator on a YM10 membrane (10 kD MWCO) (Millipore/Amicon,Bedford, Mass.) to a nominal volume (20-30 ml). The pH was adjusted to7.0 then the sample was loaded onto either a 0.8 ml Poros AL column thathad about 3 mg of zalpha11CFLAG soluble receptor or one with about 10 mgof IL-21-Fc4 fusion soluble receptor immobilized on the resin at 1ml/min on a BioCad SPRINT. The column was then washed with at least 20CV of 0.3 M NaCl/PBS(Gibco BRL)/0.01 M MES at 10 ml/min. The column wasthen rapid eluted with a 600 μl injection of 0.1 M glycine (AminoaceticAcid; Glycocol, Spectrum, Gardena, Calif.) pH 2.5 at a flow rate of 10ml/min with PBS on a BioCAD SPRINT. The 1 ml fractions were collectedfor 6 seconds each and immediately pH neutralized with 55 μl of 2 M TRIS(Tris (Hydroxymethyl) Aminomethane, EM Science, Gibbstown, N.J.) pH 8.8.The absorbence at 280 and 215 nM were monitored over the entirechromatography. Fractions were analyzed as above.

Peak fractions were pooled then concentrated via diafiltration in astirred cell concentrator on a YM10 membrane (10 kD MWCO)(Millipore/Amicon, Bedford, Mass.) to 1-2 ml. The sample was then loadedon an appropriate Sephacryl S-200 (Pharmacia, Uppsala, Sweden) highresolution size exclusion column equilibrated in PBS (Gibco BRL) at anoptimal flow rate; fractions were collected over the entirechromatography and absorbence at 280 and 215 nM were monitored.Fractions were analyzed as above.

The fractions of interest were pooled and concentrated with 5 Kd MWCOMillipore centrifugal spin concentrators to a nominal volume. The finalproduct was then analyzed by SDS-PAGE Coomassie (Sigma, St. Louis, Mo.),Western immunological blotting, N-terminal sequencing, Amino AcidAnalysis, and BCA (Pierce, Rockford, Ill.) for protein purity andconcentration. Bulk protein stored as described above.

Example 6 IL-21-dependent Proliferation of B-cell Cells StimulatedAnti-CD40 or Anti-IgM

A. Purification of Human B Cells

A vial containing 1×10⁸ frozen, apheresed human peripheral bloodmononuclear cells (PBMCs) was quickly thawed in a 37° C. water bath andresuspended in 25 ml B cell medium (RPMI Medium 1640 (JRH Biosciences.Lenexa, Kans.), 10% Heat inactivated fetal bovine serum, 5% L-glutamine,5% Pen/Strep) (Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.).Cells were tested for viability using Trypan Blue (Gibco BRL). Tenmilliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc.,Piscataway, N.J.) was layered under the cell suspension and spun for 30minutes at 1800 rpm and allowed to stop with the brake off. Theinterface was then removed and transferred to a fresh 50 ml Falcon tube,brought up to a final volume of 40 ml with PBS and spun for 10 minutesat 1200 rpm with the brake on. The viability of the isolated cells wasagain tested using Trypan Blue. Alternately fresh drawn human blood wasdiluted 1:1 with PBS (Gibco BRL) and layered over Ficoll/Hypaque Plus(Pharmacia), spun and washed as above. Cells isolated from either freshor frozen sources gave equivalent results.

B cells were purified from the Ficoll floated peripheral blood cells ofnormal human donors (above) with anti-CD19 magnetic beads (MiltenyiBiotec, Auburn, Calif.) following the manufacturer's instructions. Thepurity of the resulting preparations was monitored by flow cytometricanalysis with anti-CD22 FITC Ab (Pharmingen, San Diego, Calif.). B cellpreparations were typically >90% pure.

B. Purification of Murine B Cells

A suspension of murine splenocytes was prepared by teasing adult C57Bl/6mouse (Charles River Laboratories, Wilmington, Mass.) spleens apart withbent needles in B cell medium. RBCs were removed by hypotonic lysis.CD43 positive cells were removed with CD43 magnetic beads (MiltenyiBiotec) following the manufacturer's instructions. The purity of theresulting preparations was monitored by flow cytometric analysis withanti-CD45R FITC Ab (Pharmingen). B cell preparations were typically >90%pure.

C. Proliferation of Anti-CD40-stimulated B-Cells in the Presence ofHuman or Murine IL-21

The B cells from either the human or mouse source were resuspended at afinal concentration of 1×10⁶ cells/ml in B cell medium and plated at 100μl/well in a 96 well U bottom plate (Falcon, VWR) containing variousstimulation conditions to bring the final volume to 200 μl/well. Foranti-CD40 stimulation human cultures were supplemented with 1 μg/mlanti-human CD40 (Genzyme, Cambridge, Mass.) and mouse cultures weresupplemented with 1 μg/ml anti-murine CD40 (Serotec, UK). Human ormurine IL-21 was added at dilutions ranging from 1 μg/ml-100 ng/ml. Thespecificity of the effect of IL-21 was confirmed by inhibition of IL-21with 25 mg/ml soluble human IL-21 CEE. All treatments were performed intriplicate. The cells were then incubated at 37° C. in a humidifiedincubator for 120 hours (human) or 72 hours (mouse). Sixteen hours priorto harvesting, 1 μCi ³H-thymidine (Amersham, Piscataway, N.J.) was addedto all wells to assess whether the B-cells had proliferated. The cellswere harvested into a 96 well filter plate (UniFilter GF/C, Packard,Meriden, Conn.) using a cell harvester (Packard) and collected accordingto manufacturer's instructions. The plates were dried at 55° C. for20-30 minutes and the bottom of the wells were sealed with an opaqueplate sealer. To each well was added 0.25 ml of scintillation fluid(Microscint-O, Packard) and the plate was read using a TopCountMicroplate Scintillation Counter (Packard).

Incubation with IL-21 at concentrations of 3 ng/ml or more enhanced theproliferation induced by soluble anti-CD40 in a dose dependent manner inboth murine and human B cells by as much as 30 fold. The murine andhuman B cells responded equally as well to their respective IL-21species. In both species, the stimulation was specific to IL-21, as itwas reversed by the presence of soluble IL-21 receptor in the culture.

D. Proliferation of Anti-IgM-stimulated B-Cells in the Presence of Humanor Murine IL-21

The B cells from either human or mouse source as described above wereplated as described above. For anti-IgM stimulation of human cells theplates were pre-coated overnight with 10 mg/ml F(ab′)₂ anti-human IgMAbs (Southern Biotech Associates, Birmingham, Ala.) and washed withsterile media just prior to use. The cultures were supplemented with0-10 ng/ml hu rIL-4 (R&D Systems, Minneapolis, Minn. For anti-IgMstimulation of murine cells soluble anti-IgM (Biosource, Camarillo,Calif.) was added to the cultures at 10 mg/ml. To each of the precedinganti-IgM/IL-4 conditions, human or murine IL-21 was added at dilutionsranging from 1 pg/ml-100 ng/ml as described above. The specificity ofthe effect of IL-21 was confirmed by inhibition with soluble human IL-21receptor. All treatments were performed in triplicate. The cells wereincubated, labeled with ³H-thymidine, harvested, and analyzed.

Incubation with IL-21 at concentrations of 0.3 ng/ml or more inhibitedthe proliferation induced by insoluble anti-IgM (mouse) or anti-IgM andIL-4 (human) in a dose-dependent manner. This inhibition was specific toIL-21, as it was reversed by the presence of soluble IL-21 receptor inthe culture.

Example 7 ¹²⁵I-labeled Human IL-21 Binding Study in Cell Lines

25 micrograms of purified human IL-21 was labeled with 2 mCI ¹²⁵I usingIODO-BEADS® (Pierce, Rockford Ill.), according to manufacturer'sinstructions. This labeled protein was used to asses human IL-21 bindingto human Raji cells (ATCC No. CCL-86), using binding to wild-type murineBaF3 cells, and BaF3 cells transfected with IL-21 receptor (BaF3/hIL-21cells) as controls. IL-21 binding to BaF3/hIL-21 cells was expected(positive control), while no binding to wild-type BaF3 cells wasexpected (negative control), based on proliferation assay. About 5×10⁵Raji cells/well, 1×10⁶ BaF3/hIL-21 and 1×10⁶ BaF3 cells cells/well, wereeach plated in 96-well plates. Ten ng/ml of labeled human IL-21 wasadded in duplicate to wells, with a dilution series of unlabeled humanIL-21 competitor added from 250 fold molar excess in 1:4 dilutions downto 0.061 fold molar excess. Each point was run in duplicate. After thelabeled human IL-21 was added to wells, it was allowed to incubate at 4°C. for 2 h to allow for binding of IL-21 to the cells. The cells werethen washed 3× in binding buffer (RPMI-1710 (JRH Bioscience) with 1% BSA(Sigma)), and counted on the COBRA II AUTO-GAMMA gamma counter (PackardInstrument Company, Meriden, Conn.).

Binding of the labeled IL-21 to cells was evident in the Raji and theBaF3/hIL-21 receptor cells. In addition, for Raji cells, an average 250fold molar excess of unlabeled IL-21 decreased binding 3 fold in thepresence of a non-specific unlabeled competitor (Interferon Gamma fromR&D Systems, Minneapolis, Minn.), and 3.7 fold relative to nocompetitor. Competition was observed in a dose dependent fashion for thespecific unlabeled competitor, human IL-21. Thus, the IL-21 binding toRaji cells was specific. Similarly, for positive control BaF3/IL-21receptor cells, the 250 fold molar excess of unlabeled IL-21 decreasedbinding 2 fold relative to the non-specific competitor and 3.06 foldrelative to no competitor. Thus, the IL-21 binding to BaF3/IL-21receptor cells also was specific. No compeatable binding was observedwith the wild-type BaF3 cells. Thus, the IL-21 was shown to bindspecifically to Raji cells, and to BaF3/hIl-21 cells, but not to thenegative control BaF3 cells.

Example 8 IL-21 Receptor Expression on Human Blood Cells

A. Preparation and Culture of Human Peripheral Blood Cells

Fresh drawn human blood was diluted 1:1 with PBS (GIBCO BRL) and layeredover Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway,N.J.) and spun for 30 minutes at 1800 rpm and allowed to stop with thebrake off. The interface layer was removed and transferred to a fresh 50ml Falcon tube (Falcon, VWR, Seattle, Wash.), brought up to a finalvolume of 40 ml with PBS and spun for 10 minutes at 1200 rpm with thebrake on. The viability of the isolated cells was tested using TrypanBlue (GIBCO BRL) and the cells were resuspended at a final concentrationof 1×10⁶ cells/ml cell medium (RPMI Medium 1640, 10% Heat inactivatedfetal bovine serum, 5% L-glutamine, 5% Pen/Strep) (GIBCO BRL).

Cells were cultured in 6 well plates (Falcon, VWR) for 0, 4 or 24 hourswith a variety of different stimuli described below. Anti-IgM, anti-CD40and anti-CD3 stimulation were done as in Example 44 and Example 42.Phorbol myristate acetate (PMA) and ionomycin (Sigma, St. Louis, Mo.)(Example 5C) were added to appropriate wells at 10 ng/ml and 0.5 mg/mlrespectively. The cells were incubated at 37° C. in a humidifiedincubator for various times.

B. Antibody Staining and Analysis

Cells were collected out of the plates, washed and resuspended in icecold staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) ata concentration of about ten million cells per milliliter. Blocking ofFc receptor and non-specific binding of antibodies to the cells wasachieved by adding 10% normal goat serum (Gemini Bioproducts, Woodland,Calif.) and 10% normal human serum (Ultraserum, Gemini) to the cellsuspension. Aliquots of the cell suspensions were mixed with a FITClabeled monoclonal antibody against one of the lineage markers CD3, CD19or CD14 (PharMingen, La Jolla, Calif.) and a biotinylated monoclonalantibody against the human IL-21 receptor. Staining specificity wasdetermined by competition using IL-21CEE soluble receptor at a ten foldmass excess. After incubation on ice for 60 minutes the cells werewashed twice with ice cold staining media and resuspended in 50 mlstaining media containing streptavidin-PE (Caltag, Burlingame, Calif.).After a 30 minute incubation on ice, the cells were washed twice withice cold wash buffer (PBS, 1% fetal bovine serum, 0.1% sodium azide) andresuspended in wash buffer containing 1 mg/ml 7-AAD (Molecular Probes,Eugene, Oreg.) as a viability marker. Flow data was acquired on livingcells using a FACSCalibur flow cytometer (BD Immunocytometry Systems,San Jose, Calif.). Both acquisition and analysis were performed usingCellQuest software (BD Immunocytometry Systems).

Results of staining by anti-IL-21 antibody showed that the human IL-21receptor is expressed on human peripheral blood cells expressing eitherCD3, CD19 or CD14. Staining on CD3 and CD19 cells was specific, asevidenced by absolute competion with the IL-21 soluble receptor.Staining on CD14 cells showed some specificity for the Ligand, asevidenced by partial competion with the soluble receptor. Activation ofeither T cells with anti-CD3 or B cells with anti-CD40 resulted in anincreased level of cell surface IL-21 receptor at 24 hours. No increasein the level of expression of IL-21 receptor was seen at 4 hours withany stimulus on either cell population. Treatment of the cells withIL-21 resulted in a decrease of IL-21 receptor staining on CD3 positiveand CD19 positive cells but not CD14 positive cells at both 4 and 24hours.

Example 9 Preparation of IL-21 Antagonists

Construction of IL-21 Mutants

A total of seven constructs (of which three are shown herein) were madeusing a Stratagene QuikChange Mutagenesis kit (Catalog 200518). 100 ng.of template was used with 125 ng. of each of the sense and antisenseoligonucleotides. 25 cycles each of 94° C. for 1 min, 55° C. for 1 min.and 68° C. for 25 min. were performed for each separate reaction. Thefollowing oligonucleotide and template pairs were used:

Mutations were incorporated into the IL-21 coding sequence included inthe baculovirus expression vector BVpIL-21 using site directedmutagenesis. 100 ng of template was combined with 125 ng each ofantisense and sense oligonucleotides as shown in the table below.Thermally stable DNA polymerase was added and DNA containing the desiredmutations was synthesized during 25 thermal cycles of 94° C. for 1minute, 55° C. for 1 minute and 68° C. for 25 minutes.

Construct Oligos(sense/antisense) Template I148Stop zc27885/zc27884 (SEQID. NOS: 25 and 26) BV palpha11L Q145D/I148D zc37198/zc37199 (SEQ ID.NOS: 27 and 28) BV palpha11L-Q145D Q145D/I148Stop zc37200/zc37203 (SEQID. NOS: 29 and 30) BV palpha11L-Q145D

Following synthesis the parental, non-mutated DNA was removed bydigestion with the restriction enzyme DPN1, and the mutated DNA wastransformed into ElectroCompetent DH10B (Life Technologies). Thetransformed cells were plated for selection onto LB plates containing100 μg/mL ampicillin. Clones were analyzed by DNA sequencing and 1 μl ofthe positive clone was transformed into 20 μl DH10Bac Max Efficiencycompetent cells (Life Technologies) according to manufacturer'sinstructions.

The resulting mutants were designated Q153D;I156D as shown in SEQ ID NO:5 (nucleotide sequence) and SEQ ID NO: 6 (amino acid sequence), andI156ST;Q153D as shown in SEQ ID NO: 3 (nucleotide sequence), and SEQ IDNO: 4 (amino acid sequence.)

Example 10

A. Identification of Antagonist Activity in IL-21 Mutants

IL-21 mutant clones were expressed in baculovirus as described inExample 5. Insect cells expressing wild type human IL-21 and each of theD-helix mutants was cultured in serum free conditions. Culture media wascollected and the concentration of IL-21 or mutant IL-21 was determinedby western blot analysis using anti-IL-21 rabbit polyclonal antibody,D1048, for detection.

B. Human IL-21 Binding Studies in Cell Lines

Each mutant protein was evaluated for its ability to inhibit binding of¹²⁵I-IL-21 to receptors expressed on the surface of BHK cellstransfected with IL-21 receptor and IL-2Rγ. An IC₅₀ for inhibition of¹²⁵I-IL-21 binding was determined for each protein. Receptor activationby each protein is expressed as an EC₅₀ determined by the ability tosupport the growth of a IL-21 dependent BaF3 cell line expressingIL-21Rα and IL2Rγ.

Human IL-21 was iodinated using IODO-BEADS® (Pierce) according tomanufacturer's directions. For competition binding assays, 250 pM¹²⁵I-IL-21 was used increasing concentrations of competitor proteins.Cells, in 24 well tissue culture dishes, were incubated in 250 μl ofbinding buffer (RPMI1640 (GIBCO-BRL), 20 mM HEPES, pH7.4, 1 mg/mL BSA(Sigma)) containing ¹²⁵I-IL-21, with or without inhibitors, for 2 hoursat 4° C. Unbound ligand was removed by by three washes with ice coldbinding buffer. The cells were then extracted with PBS, 1% Triton-X100(Sigma) and the extracts counted in a gamma counter (Packard). Analysiswas done using GraphPad PRISM® (GraphPad software, Inc., San Diego,Calif.)

Transfected BaF3 cells expressing human IL-21 receptor are washed 3×with RPMI/10% FBS to remove trace amounts of IL-3. BaF3 cells are thenplated at 7500 cells/well in 96 well dish and cultured for three days inthe absence or presence of IL-21 or IL-21 mutants at concentrationsranging from 1 ng/mL to 1 μg/mL. After three days the cultures areassayed for viable cells using AlamarBlue (Alamar, Inc.) according tomanufacturer's instructions. EC50's for IL-21 and IL-21 mutants arecalculated using GraphPad PRISM® (GraphPad software, Inc.).

TABLE 6 Sample IC₅₀ (nM) EC₅₀ (nM) hIL-21 10  10 I156STOP 20 100Q153D;I156D 10 ND I156ST;Q153D 10 ND IC₅₀ and EC₅₀ are expressed as nMand ND represents no activity detected.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide molecule comprising a nucleotide sequenceas shown in SEQ ID NO: 3 or SEQ ID NO:
 5. 2. An isolated polynucleotidemolecule encoding an IL-21 antagonist polypeptide said polypeptidecomprising an amino acid sequence as shown as in SEQ ID NO: 4 or SEQ IDNO:
 6. 3. An expression vector, comprising the isolated nucleic acidmolecule of claim 1, a transcription promoter, and a transcriptionterminator, wherein the promoter is operably linked with the nucleicacid molecule, and wherein the nucleic acid molecule is operably linkedwith the transcription terminator.
 4. A recombinant host cell comprisingthe expression vector of claim 3, wherein the host cell is selected fromthe group consisting of bacterium, yeast cell, fungal cell, insect cell,mammalian cell, and plant cell.
 5. A method of producing a polypeptide,the method comprising the step of culturing recombinant host cells thatcomprise the expression vector of claim 3, and that produce thepolypeptide.